Salts, prodrugs and polymorphs of fab i inhibitors

ABSTRACT

In part, the present invention is directed to antibacterial compounds and salts thereof.

This application is a continuation application of U.S. Ser. No.12/032,001, filed Feb. 15, 2008, which claims priority to U.S. Ser. No.60/890,319, filed Feb. 16, 2007, each of which is hereby incorporated byreference in its entirety.

INTRODUCTION

Infections caused by or related to bacteria are a major cause of humanillness worldwide, and the frequency of resistance to standardantibiotics has risen dramatically over the last decade. Hence, thereexists an unmet medical need and demand for new agents acting againstbacterial targets.

Examples of potential bacterial targets are those enzymes involved infatty acid biosynthesis. While the overall pathway of saturated fattyacid biosynthesis is similar in all organisms, the fatty acid synthase(FAS) systems vary considerably with respect to their structuralorganization. It is believed that vertebrates and yeast possess a FAS inwhich all the enzymatic activities are encoded on one or two polypeptidechains, respectively, and the acyl carrier protein (ACP) is an integralpart of the complex. In contrast, in bacterial FAS, it is known thateach of the reactions is catalyzed by a distinct, mono-functional enzymeand the ACP is a discrete protein. Therefore, it may be possible toachieve selective inhibition of the bacterial system by appropriateagents.

One such potential bacterial target is the FabI protein. FabI(previously designated EnvM) is believed to function as an enoyl-ACPreductase in the final step of the four reactions involved in each cycleof bacterial fatty acid biosynthesis. It is believed that in thispathway, the first step is catalyzed by β-ketoacyl-ACP synthase, whichcondenses malonyl-ACP with acetyl-CoA (FabH, synthase III). It isbelieved that in subsequent rounds, malonyl-ACP is condensed with thegrowing-chain acyl-ACP (FabB and FabF, synthases I and II,respectively). The second step in the elongation cycle is thought to beketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG).Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA orFabZ) leads to trans-2-enoyl-ACP. Finaly, in step four,trans-2-enoyl-ACP is converted to acyl-ACP by an NADH (orNADPH)-dependent enoyl-ACP reductase (Fab I). Further rounds of thiscycle, adding two carbon atoms per cycle, would eventually lead topalmitoyl-ACP (16C), where upon the cycle is stopped largely due tofeedback inhibition of Fab I by palmitoyl-ACP. Thus, Fab I is believedto be a major biosynthetic enzyme and is a key regulatory point in theoverall synthetic pathway of bacterial fatty acid biosynthesis.

In some bacteria the final step of fatty acid biosynthes is catalyzed byFab I only, in others by FabK, an NADH and FMN dependent reductase,still others utilize both FabI and FabK. The present invention provides,in part, compounds and compositions with FabI inhibiting properties.

SUMMARY

In part, the present invention is directed towards salts and polymorphsof compounds with FabI inhibiting properties as well as other enzymes.Other uses for the subject compounds and compositions will be readilydiscernable to those of skill in the art.

Also disclosed herein are methods and/or synthetic routes for largescale preparation of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand its toluene sulfonic acid salt forms.

In an embodiment, this disclosure provides for a composition that may besubstantially free of palladium and comprises(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand/or its salts, hydrates, and prodrug forms, for example, toluenesulfonic acid salt forms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a 96-well plate for salt formation after acid addition,and cooling and evaporation of the plate;

FIG. 2 depicts an XRPD spectra of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 3 depicts: A) an XRPD spectra of a hydrochloric acid salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand B) the DSC of this salt;

FIG. 4 depicts an XRPD spectra of a mesylate salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 5 depicts an XRPD spectra of benzenesulfonic acid salt from THF of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 6 depicts an XRPD spectra of benzenesulfonic salt from DCM of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 7 depicts: A) an XRPD spectra of p-toluenesulfonic salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide,B) the DSC of this salt from DCM, and C) the DSC of this salt from THF;

FIG. 8 depicts an XRPD spectra of a sulfate salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 9 depicts A) an XRPD spectra of a mesylate salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideafter ball milling and B) the DSC;

FIG. 10 depicts: A) an XRPD spectra of p-toluenesulfonic salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideafter ball milling and B) the DSC of this salt after ball milling;

FIG. 11 depicts the DSC graph of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidesulfate;

FIG. 12 depicts an XRPD spectra of a p-toluenesulfonic salt monohydrateof(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide;

FIG. 13 depicts a XRPD spectra of the anhydrous form ofp-toluenesulfonic salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide.

FIG. 14 depicts the oral efficacy of the tosylate salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidein comparision to the free base form of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidein a mouse thigh abscess model.

FIG. 15 depicts plasma time concentrations of the free base and thetosylate monohydrate salt of Compound A in a rat model.

DETAILED DESCRIPTION Introduction

The present disclosure is directed in part towards novel compoundsand/or compositions that inhibit bacterial enzymes, and methods ofmaking and using the same. In part, the disclosure is directed to salts,prodrugs, hydrates, and/or polymorphs of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide,referred to as “compound A” throughout this disclosure.

DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “FabI” is art-recognized and refers to the bacterial enzymebelieved to function as an enoyl-acyl carrier protein (ACP) reductase inthe final step of the four reactions involved in each cycle of bacterialfatty acid biosynthesis. This enzyme is believed to be widelydistributed in bacteria and plants.

The term “enzyme inhibitor” refers to any compound that prevents anenzyme from effectively carrying out its respective biochemical roles.Therefore a “FabI inhibitor” is any compound that inhibits FabI fromcarrying out its biochemical role. The amount of inhibition of theenzyme by any such compound will vary and is described herein andelsewhere.

The term “antibiotic agent” shall mean any drug that is useful intreating, preventing, or otherwise reducing the severity of anybacterial disorder, or any complications thereof, including any of theconditions, disease, or complications arising therefrom and/or describedherein. Antibiotic agents include, for example, cephalosporins,quinolones and fluoroquinolones, penicillins, penicillins and betalactamase inhibitors, carbepenems, monobactams, macrolides andlincosamines, glycopeptides, rifampin, oxazolidonones, tetracyclines,aminoglycosides, streptogramins, sulfonamides, and the like. Othergeneral categories of antibiotic agents which may be part of a subjectcomposition include those agents known to those of skill in the art asantibiotics and that qualify as (with defined terms being in quotationmarks): “drug articles” recognized in the official United StatesPharmacopoeia or official National Formulary (or any supplementthereto); “new drug” and “new animal drug” approved by the FDA of theU.S. as those terms are used in Title 21 of the United States Code; anydrug that requires approval of a government entity, in the U.S. orabroad (“approved drug”); any drug that it is necessary to obtainregulatory approval so as to comply with 21 U.S.C. §355(a) (“regulatoryapproved drug”); any agent that is or was subject to a human drugapplication under 21 U.S.C. §379(g) (“human drug”). (All references tostatutory code for this definition refer to such code as of the originalfiling date of this provisional application.) Other antibiotic agentsare disclosed herein, and are known to those of skill in the art. Incertain embodiments, the term “antibiotic agent” does not include anagent that is a FabI inhibitor, so that the combinations of the presentinvention in certain instances will include one agent that is a FabIinhibitor and another agent that is not.

The term “illness” as used herein refers to any illness caused by orrelated to infection by an organism.

The term “bacterial illness” as used herein refers to any illness causedby or related to infection by bacteria.

The term “cis” is art-recognized and refers to the arrangement of twoatoms or groups around a double bond such that the atoms or groups areon the same side of the double bond. Cis configurations are oftenlabeled as (Z) configurations.

The term “substantially the same” when used to describe X-ray powderdiffraction patterns, is meant to include patterns in which peaks arewithin a standard deviation of ±0.2 2θ.

The term “trans” is art-recognized and refers to the arrangement of twoatoms or groups around a double bond such that the atoms or groups areon the opposite sides of a double bond. Trans configurations are oftenlabeled as (E) configurations.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. Antibiotic agents andFab I/Fab K inhibitors are examples of therapeutic agents.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The term thusmeans any substance intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease or in the enhancement of desirablephysical or mental development and/or conditions in an animal or human.The phrase “therapeutically-effective amount” means that amount of sucha substance that produces some desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. Thetherapeutically effective amount of such substance will vary dependingupon the subject and disease condition being treated, the weight and ageof the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. For example, certain compositions of thepresent invention may be administered in a sufficient amount to producea at a reasonable benefit/risk ratio applicable to such treatment.

The term “meso compound” is art-recognized and refers to a chemicalcompound which has at least two chiral centers but is achiral due to aplane or point of symmetry.

The term “chiral” is art-recognized and refers to molecules which havethe property of non-superimposability of the mirror image partner, whilethe term “achiral” refers to molecules which are superimposable on theirmirror image partner. A “prochiral molecule” is a molecule which has thepotential to be converted to a chiral molecule in a particular process.

The term “stereoisomers” is art-recognized and refers to compounds whichhave identical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space. In particular,“enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another. “Diastereomers”, on theother hand, refers to stereoisomers with two or more centers ofdissymmetry and whose molecules are not mirror images of one another.

Furthermore, a “stereoselective process” is one which produces aparticular stereoisomer of a reaction product in preference to otherpossible stereoisomers of that product. An “enantioselective process” isone which favors production of one of the two possible enantiomers of areaction product.

The term “regioisomers” is art-recognized and refers to compounds whichhave the same molecular formula but differ in the connectivity of theatoms. Accordingly, a “regioselective process” is one which favors theproduction of a particular regioisomer over others, e.g., the reactionproduces a statistically significant increase in the yield of a certainregioisomer.

The term “epimers” is art-recognized and refers to molecules withidentical chemical constitution and containing more than onestereocenter, but which differ in configuration at only one of thesestereocenters.

The term “ED₅₀” is art-recognized. In certain embodiments. ED₅₀ meansthe dose of a drug which produces 50% of its maximum response or effect,or alternatively, the dose which produces a pre-determined response in50% of test subjects or preparations. The term “LD₅₀” is art-recognized.In certain embodiments, LD₅₀ means the dose of a drug which is lethal in50% of test subjects. The term “therapeutic index” is an art-recognizedterm which refers to the therapeutic index of a drug, defined asLD₅₀/ED₅₀.

The term “K_(i)” is art-recognized and refers to the dissociationconstant of the enzyme-inhibitor complex.

The term “antimicrobial” is art-recognized and refers to the ability ofthe compounds of the present invention to prevent, inhibit or destroythe growth of microbes such as bacteria, fungi, protozoa and viruses.

The term “antibacterial” is art-recognized and refers to the ability ofthe compounds of the present invention to prevent, inhibit or destroythe growth of microbes of bacteria.

The term “microbe” is art-recognized and refers to a microscopicorganism. In certain embodiments the term microbe is applied tobacteria. In other embodiments the term refers to pathogenic forms of amicroscopic organism.

The term “prodrug” is art-recognized and is intended to encompasscompounds which, under physiological conditions, are converted into theantibacterial agents of the present invention. A common method formaking a prodrug is to select moieties which are hydrolyzed underphysiological conditions to provide the desired compound. In otherembodiments, the prodrug is converted by an enzymatic activity of thehost animal or the target bacteria.

The term “aliphatic” is art-recognized and refers to a linear, branched,cyclic alkane, alkene, or alkyne. In certain embodiments, aliphaticgroups in the present invention are linear or branched and have from 1to about 20 carbon atoms.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure. The term “alkyl” is also defined to include halosubstitutedalkyls.

Moreover, the term “alkyl” (or “lower alkyl”) includes “substitutedalkyls”, which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents may include, for example, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CN and thelike. Exemplary substituted alkyls are described below. Cycloalkyls maybe further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CN, and the like.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g. an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “heteroaryl” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to 3- to about 10-membered ring structures, alternatively 3- toabout 7-membered rings, whose ring structures include one to fourheteroatoms. Heterocycles may also be polycycles. Heterocyclyl groupsinclude, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on 560 of “AdvancedInorganic Chemistry” by Cotton and Wilkinson.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate, p-toluenesulfonate, methanesulfonate, andnonafluorobutanesulfonate functional groups and molecules that containsaid groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g. which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67 s Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds that may besubstituted or unsubstituted.

The definition of each expression, e.g. lower alkyl, m, n, p and thelike, when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The term “treating” is art-recognized and refers to curing as well asameliorating at least one symptom of any condition or disease.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

A “patient,” “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, bovines, porcines, canines, felines, and rodents(e.g., mice and rats).

The term “bioavailable” is art-recognized and refers to a form of thesubject invention that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

The term “pharmaceutically-acceptable salts” is art-recognized andrefers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds, including, for example, those contained incompositions of the present invention.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof from one organ, or portion ofthe body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water, (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The terms “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized and refer to the administration of a subject composition,therapeutic or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

Contemplated equivalents of the compositions described herein includecompositions which otherwise correspond thereto, and which have the samegeneral properties thereof (such as other compositions comprisingFabI/Fab K inhibitors or salts and/or hydrates and/or prodrugs and/orpolymorphs of Compound A), wherein one or more simple variations ofsubstituents or components are made which do not adversely affect thecharacteristics of the compositions of interest. In general, thecomponents of the compositions of the present invention may be preparedby the methods illustrated in the general reaction schema and writtenprocedures as, for example, described below, or by modificationsthereof, using readily available starting materials, reagents andconventional synthesis procedures. In these reactions, it is alsopossible to make use of variants which are in themselves known, but arenot mentioned here.

FabI Inhibitors

The FabI inhibitor compounds of the present invention include thosedepicted by formula I:

wherein n is a fractional or whole number between about 0 and about 1.5inclusive; m is a integer or whole (e.g. fractional) number betweenabout 0 and about 3 inclusive; X is selected from the group consistingof H₂SO₄, HSO₃R′, HSO₃Ar, H₃PO₄, HCl, HBr, CF₃CO₂H, and Cl₃CO₂H; R′ isalkyl; and Ar is aryl. Ar may be, for example, p-toluene or benzene.

In cases wherein such inhibitors may have one or more chiral centers,unless specified, the present invention comprises each unique racemiccompound, as well as each unique nonracemic compound.

In cases in which the inhibitors have unsaturated carbon-carbon doublebonds, both the cis (Z) and trans (E) isomers are within the scope ofthis invention. In cases wherein inhibitors may exist in tautomericforms, such as keto-enol tautomers, such as

each tautomeric form is contemplated as being included within thisinvention, whether existing in equilibrium or locked in one form byappropriate substitution with R′. The meaning of any substituent at anyone occurrence is independent of its meaning, or any other substituent'smeaning, at any other occurrence.

In some embodiments, n may be a fractional or whole number between about0 and about 1.5 inclusive, for example n may be about 1. In otherembodiments, m may be a fractional or whole number between about 0 andabout 3, inclusive, for example m may be 0, 1, 2 or 3.

For example, compound A may be in a free base, anhydrous form (e.g. n is0 and m is 0 in formula I). Alternatively, hydrates of compound A arealso contemplated herein (m>0), for example, a hydrate of compound A maybe a monohydrate, (e.g. n=0 and m is 1).

Hydrates are also provided when n is about 1 or more. For example, thep-toluenesulfonic salt of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-8-naphthyridin-3-yl)acrylamidemay be provided in anhydrous (m=0) or monohydrate (m=1) form.

Polymorphs of compound A, and salts, hydrates and prodrugs are alsocontemplated herein. Such polymorphs can be produced by e.g. usingcrystallization conditions to isolate a free-base and salt forms and/orby ball-milling such forms.

Also included in the antibiotic compounds of the present invention areprodrugs of the FabI inhibitors. The FabI inhibitor compounds of thepresent invention include those depicted by A compound of formula II:

wherein R is a labile modifying group that is capable of releasing thecompound(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidein vivo. The pharmaceutically acceptable salts of Formula II are alsocontemplated. In some embodiments R may be for example selected from thegroup consisting of —CO₂R′, —CH₂OC(O)R′, —PO₃Ca, —PO₃Mg, —PO₃Na₂, and—PO₃K₂, where R′ is alkyl.

Compounds disclosed herein include:(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidehydrochloride;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidehydrobromide;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidesulfate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidemethane sulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideethane sulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide2-hydroxyethanesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide4-methylbenzenesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide4-methylbenzenesulfonate monohydrate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidebenzenesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidephosphate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidetrifluoroacetate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidetrichloroacetate;(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonicacid; Calcium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;Magnesium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;Disodium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;and Dipotassium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;(Z)—N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand rotamers thereof.

Cis-trans amide rotamers of compound A and salts, hydrates or prodrugsthereof are also contemplated. For example, at ambient temperature, anamide bond may not have any free rotation. At an elevated temperature,e.g. about 70 to about 10° C. an amide molecule may establish trans andcis rotameric equilibrium. At these temperature, free rotation aroundthe amide can occur, and ¹H NMR spectrum may sharpen. Exemplary cisoidand transoid rotamers are shown by the formula III and IV:

Cis-isomers of compound A or salts, hydrates, or prodrugs thereof isalso contemplated, for example, as shown in formula V. Pharmaceuticalsalts, hydrates or prodrugs of cis-isomers are also contemplated.

R₁ may be H or alkyl, e.g., methyl, R₂ may be H or alkyl, and X may be Oor S.

Reaction Methods

Methods I to III are directed to chemistry involved in the synthesis ofsalts and/or hydrates of compound A. One of skill in the art wouldunderstand that the disclosed salts can be made in a variety of waysthat may differ from the exemplary schemes described below. Acidaddition salts of the compounds of formula I can be prepared in astandard manner in a suitable solvent from the parent compound and anexcess of an acid, such as hydrochloric, hydrobromic, hydrofluoric,sulfuric, phosphoric, acetic, trifluoroacetic, maleic, citric, succinicor methanesulfonic. Certain of the compounds form inner salts orzwitterions which may be acceptable. Cationic salts may be prepared bytreating the parent compound with an excess of an alkaline reagent, suchas a hydroxide, carbonate or alkoxide, containing the appropriatecation; or with an appropriate organic amine. Cations such as Li⁺, Na⁺,K⁺, Ca⁺⁺, Mg⁺⁺ and NH₄ ⁺ are some non-limiting examples of cationspresent.

Method I

General Method of Salt Formation 1: Combinatorial Screen

A matrix of 10 acids (maleic acid, p-toluenesulfonic acid,benzenesulfonic acid, methansulfonic acid, trichloroacetic acid,trifluoroacetic acid, hydrobromic acid, hydrochloric acid, sulfuricacid, and phosphoric acid) and six solvents (dioxane, THF, DMF, AcOH,DCM, DMA) can be laid out in a e.g. 96 well plate with the counter ionsoccupying the first 10 columns and the solvents the first six rows. Eachwell can be charged with e.g. 4.66×10⁻⁶ mol of the(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideby combinatorially dispensing 500 μL of a 0.009 molar stock solution ofcompound A dissolved in solvent, for example, dichloromethane (DCM). Thecontent of each well can then be concentrated to dryness under a slowstream of nitrogen using a 96 well plate nitrogen manifold.

The residues can be dissolve, for example, in 500 μL of e.g. 6 solvents(dioxane, THF, DMF, AcOH, DCM, DMA). An extra 250 μL can be added to thewells for dioxane and THF. The plate is placed in a heated block at 55°C. on top of an orbital shaker and shaken for 10 minutes to ensuredissolution. Each well is then charged with 39 μL of a 0.126 M solutionof the acid in dioxane corresponding to 1.05 equivalents of each of the10 acids. The plate is then placed back onto the orbital shaker andcooled to room temperature at a rate of 20° C./hour, after 3 hours thesolvents were removed under a stream of nitrogen.

Determination of crystallinity can be performed on solid-containingwells by dispersing the solids in mineral oil and using a polarizedlight microscope at 100× and 400×. Samples which contain particles withdistinct birefringence and extinction positions are typically identifiedas crystalline. Those which have particles that exhibit birefringenceand extinction positions but also had a significant amount of particleswhich were not observed to reflect light can be typically described aspartially crystalline. Amorphous solids are typically those which do nothave significant amount of particles that reflected polarized lightwhile deliquescent material becomes liquid-like or softened duringanalysis.

Table 1 summarizes representative solvents and amounts used for thegeneration of HBr, sulfuric acid, methane sulfonic acid (MsOH), benzenesulfonic acid and p-toluene sulfonic acid salts of compound A but may beapplied to any of the combinations described herein.

TABLE 1 Compound A Heptane amount Amount Amount Recovery (mg) Solvent(mL) Acid (mL) (mg) 19 THF 8 HBr 3 0.007 20 DCM 4 HBr 3 0.016 19 THF 8sulfuric 3 0.004 19 THF 8 MsOH 5 0.015 20 DCM 4 MsOH 2 0.019 20 THF 8benzene- 4 0.007 sulfonic 20 DCM 5 benzene- 3 0.015 sulfonic 20 THF 8p-toluene- 4 0.004 sulfonic 20 DCM 5 p-toluene- 3 0.016 sulfonic

Method II

General Method of Salt Formation II: Intermediate Scale

Variant A

This method employs combinations of counter-ion and solvent. Vials werecharged with between 19-21 mg of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand are followed by one of the following solvents: DCM 2 mL, THF 10 mL,AcOH 4.5 mL, and DMA 3-4 mL. These vials are heated (DCM 32° C., THF 58°C., AcOH and DMA 90° C.) with stirring to ensure dissolution. Each vialis then charged with between 425-465 μL of a 0.126 M solution of theacid in dioxane corresponding to 1.05 equivalents of each of the acids.

The temperature is held at the dissolution temperature for 10 minutesand is then ramped down to room temperature at a rate of 20° C./hour.Typically, upon reaching room temperature the vials do not show anyprecipitation at this stage. The vials are allowed to evaporate toinduce precipitation. Upon losing about one quarter of the volume theTHF examples typically start to precipitate. The solids are thengenerally collected by filtration. In some cases the (eg. some DCMexamples) most of the volume of solvent can evaporate beforeprecipitation occurs and can not be filtered. In other situations, thevial contents may have to be concentrated before crystallization occurs.All solid products can be analyzed directly by XRPD and DSC.

Variant B

In a variant of general method II vials are charged with between 19-21mg of the API were treated with one of the above solvents (i.e. DCM orTHF). These vials are heated (DCM 32° C., THF 58° C.) with stirring toensure dissolution. Each vial is then charged with between 425 to 465 μLof a 0.126 M solution of the acid in dioxane corresponding to 1.05equivalents of each acid. The temperature is held (DCM 32° C., THF 58°C.) for 10 minutes before the introduction of an anti-solvent such asheptane. Heptane can be added until the solution became turbid, 3 mL forTHF experiment and 1.5 mL for DCM experiment. After addition the vialsare allowed to cool to room temperate at a rate of 20° C./hour.Precipitation typically occurs during the cooling phase and the solidscan be collected by filtration and dried in vacuo at 50° C. and 30inches of Hg.

Method III

General Method of Salt Formation III: Scale-Up

Scale-up reactions can be carried out on, for example, 1 gram ofmaterial. Table 2 summarizes typical amounts of solvents and reagentsused for exemplary scale up reactions although variations in solvent andCompound A amount can be employed.

TABLE 2 Amount Anti- Compound A solvent of 1M solvent amnt amnt solutionamnt Yield (g) Solvent (mL) Acid (mL) (mL) % 1.000 THF 350 p-TSA 2.8 14477.8 1.016 THF 350 sulfuric 2.82 34 83.7 1.000 DCM 200 MsOH 2.8 150 84.4

Compound A is dissolved in the appropriate amount of solvent withheating and stirring. To this is added the acid solution at for example1M concentration, and the delivery solvent is matched with the reactionsolvent. After stirring for 5 minutes the anti-solvent (typically ahydrocarbon e.g. heptane) is added. Enough anti-solvent is added to forma turbid solution that typically clears up after a few seconds. Thereactions are heated back to the reaction temperature if any coolingoccurred during the anti-solvent addition. The temperatures are thenramped down to room temperature at a rate of about 20° C./hour. Thesolids are then collected by filtration and dried in vacuo (55° C. and30 inches of Hg).

Method IV

Phosphonate Prodrugs

Processes for the prepartion of Compound A phosphonic acid and its saltsare outlined in Scheme I. For example. Compound A is reacted with baseand a source of di-(arylmethyl)-phosphoro radical such as, for example,dibenzylphosphoro chloride or tetrabenzylpyrophosphate to obtain thediarylmethyl phosphonate, attached at the naphthyridinyl 8-nitrogen ofCompound A as shown in Scheme I. Arylmethyl groups attached to thephosphate, such as benzyl, can generally be removed by hydrogenolysisconditions, such as hydrogen gas with palladium on carbon catalyst, inthe presence of a metallic base, such as potassium carbonate, or anamine base, such as triethyl amine, to yield the correspondingphosphonate salts.

Alternatively, Compound A can be reacted with, for example, a base and asource of diaryl phosphate radical such as diphenylphosphoro chloride toobtain the diaryl phosphonate of Compound A attached at thenaphthyridinyl 8-nitrogen of Compound A as shown in Scheme I. Arylgroups attached to the phosphate, such as phenyl, are generally reactedunder basic conditions, such as sodium hydroxide to yield thecorresponding disodium salt of the depicted phosphonate.

When R is a small alkyl group (e.g. 1-4 carbons) trimethyl silyl iodide(TMSiI), followed by an aqueous quench may be used to generate thephosphonate of Compound A.

The phosphonate of Compound A may also be formed by reacting Compound Awith phosphoric anhydride under elevated temperature and using asubsequent treatment with moist organic solvent, such as for example,ether.

The number of counter ions, n, in the phosphonate product may bedependent on the charge resident on the counter ion, M+. By way ofillustration for a calcium salt (Ca²⁺) one counter ion would be found.Alternatively for a tertiary amine counter ion such as triethylamine(Et₃HN⁺) or a monovalent metal such as sodium (Na⁺) or potassium (K⁺),two counter ions are required.

Method V:

Large scale preparation of(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand its toluene sulfonic acid salt forms.

Processes for the prepartion of Compound A and its salts suitable forlarge scale processing are outlined in Scheme II. Exemplification ofsuch large scale processes are provided in the below Examples 10-11. Thelarge scale processes disclosed herein result in a final product thatmay be substantially free of palladium. A composition, comprising(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideand/or its salts, hydrates, and/or prodrug forms is contemplated that issubstantially free of palladium, for example, has less than about 50ppm, less than about 30 ppm, less than about 20 ppm, less than about 10ppm of palladium. For example, such a composition may have about 0.01 toabout 50 ppm of palladium, about 0.01 to about 20 ppm, about 0.01 toabout 10 ppm of pallaiduim. Also contemplated herein are compositionscomprising less than about 100 ppm of heavy metals, e.g. about 0.01 ppmto about 100 pm of heavy metals.

The large scale processes disclosed herein (e.g. processes for makinge.g. about 0.5 kg or more of material include a large scale process forpreparing substantially palladium free(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidecomprising contacting a suspension comprising(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidewith an organic base, for example N,N′-diisopropylethylamine (DIPEA);heating the suspension; and filtering the suspension to retrieve(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide.

In some embodiments, the disclosed compounds and/or compostions may besubject to further processing, for example ball milling. In someembodiments, ball milling of a disclosed compound may result in adifferent. e.g. unique, polymorph of that compound. Ball milling may beperformed in various ways as known to those skilled in the art.

For example, ball milling may be conducted by drying the solid compoundat, for example, about 50° C. in a vacuum oven for, e.g. about 48 h. Thesolid can then be milled for example in 12-g batches using a FritschPulverisette 6-ball mill in a 240-mL bowl with 150 agate balls at 200rpm for 1 min, and then blended in a large beaker.

Other exemplary balling milling procedures that result in particle sizereduction include the use of fluid energy jet mills. In this procedure,the material may be manually fed into a hopper placed on top of a feedtray. The material is drawn into a confined, circular chamber by way ofpressurized gas and is suspended in a high velocity fluid stream in themilling chamber. The mill operates on the principle of impact andattrition due to the high velocity collisions between particlessuspended within the gas stream, causing them to breakdown into smallerparticles.

Centrifugal force causes large, heavy particles to separate from smallerand lighter particles. The smaller particles are carried in the fluidstream towards the center of the milling chamber, where they aredischarged into a filter bag. The larger particles are thrown outwardwhere they re-circulate and re-collide, causing them to breakdown.Typically, operational parameters are monitored and documentedthroughout this process.

The disclosed compounds can be characterized by X-ray powderdiffractometry (XRPD). An XRPD spectrum may be obtained with ameasurement error depending on measurement conditions. In particular,intensities in a XRPD may fluctuate depending on measurement conditions.Therefore, it should be understood that the compounds providing any XRPDspectra substantially the same as the disclosed spectra fall within thescope of the present invention. Those skilled in the art can readilyjudge the substantial identity of XRPD spectra.

Generally, a measurement error of diffraction angle for a X-ray powderdiffractometry is about 5% or less, and such degree of a measurementerror should be taken into account as to diffraction angles. Forexample, the diffraction angles may be reported with a measurement errorof ±1°, ±2°, ±3°, or ±5° 2θ.

Method VI

Cis-Isomers

Scheme III shows the conversion of trans isomer to cis isomer byphotochemical equilibration.

Preparative normal phase high performance liquid chromatography orreverse phase high performance chromatography can isolate the finalproduct.

Toxicology of Compounds

Acute toxicity can be assessed using increasing doses in mice androdents. Exploratory acute toxicity in mice and/or rats after singledose may be undertaken to begin estimation of the therapeutic window ofinhibitors and to identify the potential target organis of toxicity. Ascandidate selection nears, these studies may provide guidance for theselection of proper doses in multi-dose studies, as well as establishany species specific differences in toxicities. These studies may becombined with routine PK measurements to assure proper dosages wereachieved. Generally 3-4 doses will be chosen that are estimated to spana range having no effect through to higher doses that cause major toxic,but non-lethal, effects. Animals will be observed for effects on bodyweight, behavior and food consumption, and after euthanasia, hematology,blood chemistry, urinalysis, organ weight, gross pathology andhistopathology will be undertaken.

Resistance Frequencies and Mechanisms of Compounds

In vitro resistance frequencies in bacteria of interest can be estimatedfor compounds of formula I. Experiments can determine whether resistantisolates arise when challenged to grow on solid media at 1×, 2× and4×MIC concentrations. For example with respect to S. aureus or E. Coli,the experiments may use several recent clinical isolates ofmethicillin-sensitive and methicillin-resistant S. aureus and alaboratory strain of E. coli with acrA efflux pump defect. In addition,experiments may use several characterized triclosan-resistant S. aureusstrains. The MICs of resistant strains isolated in this manner can thenbe determined. Subsequent experiments can determine whether resistantstrains arise after serial passage of the strains in 0.5×MICconcentrations of each lead compound.

Mechanism of resistance may be determined in S. aureus laboratorystrain. RN450 and in an E. coli laboratory strain carrying an acrAefflux pump mutation. Both high dose challenge (4×MIC) and sub-MICserial passage may be used to obtain spontaneously arising resistantisolates. If no isolates are obtained with reasonable frequencies,chemical and physical mutagenesis methods can be used to obtainresistant isolates. The fabI gene from the chromosome of resistantisolates may be PCR amplified, then may be sequenced to determinewhether changes in the FabI protein caused resistance. Triplicate PCRamplifications and sequences may be performed to assure that theobserved sequence changes are correct, and did not arise from PCR errorsduring amplification. Strains carrying resistance mutations outside ofthe gene of interest may be documented and saved, characterized fortheir effects on susceptibilities of other antibiotics as evidence ofpossible efflux-mediated resistance mechanisms, characterized for theirability to alter compounds characterized for their effects on theexpression of the specific mRNA and FabI protein.

Assays

Many different assay methods can be used to determine the activity ofthe compounds of the present invention. These assay methods include, forexample, the following but also include other methods known to one ofordinary skill in the art.

S. aureus FabI Enzyme Inhibition Assay (NADH).

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 50-uL assay mixtures containing 100 mM NaADA,pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol, 0.25 mMcrotonoyl CoA, 1 mM NADH, and an appropriate dilution of S. aureus FabI.Inhibitors are typically varied over the range of 0.01-10 uM. Theconsumption of NADH is monitored for 20 minutes at 30° C. by followingthe change in absorbance at 340 nm. Initial velocities are estimatedfrom an exponential fit of the non-linear progress curves represented bythe slope of the tangent at t=0 min. IC₅₀'s are estimated from a fit ofthe initial velocities to a standard, 4-parameter model and aretypically reported as the mean±S.D. of duplicate determinations.Triclosan, a commercial antibacterial agent and inhibitor of FabI, maybe included in an assay as a positive control. Compounds of thisinvention may have IC₅₀'s from about 5.0 micromolar to about 0.05micromolar.

S. Aureus FabI Enzyme Inhibition Assay (NADPH) (Modified)

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 150-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADPH, and an appropriate dilution of S.aureus FabI. Inhibitors are typically varied over the range of 0.01-10uM. The consumption of NADPH is monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities areestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s are estimatedfrom a fit of the initial velocities to a standard, 4-parameter modeland are typically reported as the mean±S.D. of duplicate determinations.Triclosan, a commercial antibacterial agent and inhibitor of FabI, iscurrently included in all assays as a positive control.

H. influenzae FabI Enzyme Inhibition Assay

Assays are carried out in half-area, 96-well microtiter plates.Compounds are evaluated in 150-uL assay mixtures containing 100 mM MES,51 mM diethanolamine, 51 mM triethanolamine, pH 6.5(MES=2-(N-morpholino)ethanesulfonic acid), 4% glycerol, 25 uMcrotonoyl-ACP, 50 uM NADH, and an appropriate dilution of H. influenzaeFabI (approximately 20 nM). Inhibitors are typically varied over therange of 0.01-10 uM. The consumption of NADH is monitored for 20 minutesat 30° C. by following the change in absorbance at 340 nm. Initialvelocities are estimated from an exponential fit of the non-linearprogress curves. IC₅₀'s are estimated from a fit of the initialvelocities to a standard, 4-parameter model, and are typically reportedas the mean±S.D. of duplicate determinations. The apparent Ki iscalculated assuming the inhibition is competitive with crotonoyl-ACP. Aproprietary lead compound is currently included in all assays as apositive control.

E. coli FabI Enzyme Inhibition Assay

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 150-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADH, and an appropriate dilution of E.coli FabI. Inhibitors are typically varied over the range of 0.01-10 uM.The consumption of NADH is monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities areestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s are estimatedfrom a fit of the initial velocities to a standard, 4-parameter modeland are typically reported as the mean±S.D. of duplicate determinations.Triclosan, a commercial antibacterial agent and inhibitor of FabI, iscurrently included in all assays as a positive control. Compounds ofthis invention have IC₅₀'s from about 100.0 micromolar to about 0.05micromolar.

Preparation and Purification of Crotonoyl-ACP

Reactions contain 5 mg/mL E. coli apo-ACP, 0.8 mM crotonoyl-CoA (Fluka),10 mM MgCl₂, and 30 uM S. pneumoniae ACP synthase in 50 mM NaHEPES, pH7.5. The mixture is gently mixed on a magnetic stirrer at 23° C. for 2hr, and the reaction is terminated by the addition of 15 mM EDTA andcooling on ice. The reaction mixture is filtered through a 0.2 micronfilter (Millipore) and applied to a MonoQ column (Pharmacia)equilibrated with 20 mM Tris-Cl, pH 7.5. The column is washed withbuffer until all non-adherent material is removed (as observed by UVdetection), and the crotonoyl-ACP is eluted with a linear gradient of 0to 400 mM NaCl.

S. Aureus FabI Enzyme Inhibition Assay Using Crotonoyl-ACP

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 100 uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-(2-acetamido)-2-iminodiacetic acid), 4% glycerol,25 uM crotonoyl-ACP, 50 uM NADPH, and an appropriate dilution of S.aureus Fab I (approximately 20 nM). Inhibitors are typically varied overthe range of 0.01-30 uM. The consumption of NADPH is monitored for 30minutes at 30° C. by following the change in absorbance at 340 nm.Initial velocities are estimated from a linear fit of the progresscurves. IC₅₀ 's are estimated from a fit of the initial velocities to astandard, 4-parameter model (Equation 1) and are typically reported asthe mean±S.D. of duplicate determinations. Compounds of this inventionin this assay have IC₅₀ 's from about 60.0 micromolar to about 0.01micromolar. The apparent Ki is calculated from Equation 2 assuming theinhibition is competitve with crotonoyl-ACP. More specifically, measuredIC₅₀ values for 24 compounds of the present invention, as provided inthe representative list above, ranged from less than about 0.02 μM toabout 25 μM with 11 of these compounds having an IC₅₀ of less than 1.

H. Pylori FabI Enzyme Inhibition Assay Using Crotonoyl-ACP

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 100 uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-(2-acetamido)-2-iminodiacetic acid), 4% glycerol,10 uM crotonoyl-ACP, 50 uM NADH, 100 mM ammonium acetate, and anappropriate dilution of H. pylori Fab I (approximately 15 nM).Inhibitors are typically varied over the range of 0.025-30 uM. Theconsumption of NADH is monitored for 30 minutes at 25° C. by followingthe change in absorbance at 340 nm. Initial velocities are estimatedfrom a linear fit of the progress curves. IC₅₀'s are estimated from afit of the initial velocities to a standard, 4-parameter model(Equation 1) and are typically reported as the mean±S.D. of duplicatedeterminations. Compounds of this invention in this assay have IC₅₀'sfrom about 60.0 micromolar to about 0.01 micromolar. The apparent K_(i)is calculated from Equation 2 assuming the inhibition is competitve withcrotonoyl-ACP.

v=Range/(1+[I]/IC50)s+Background  Equation 1:

Ki(app)=IC50/(1+[S]/Ks)  Equation 2:

S. Pneumoniae FabK Enzyme Inhibition Assay Using Crotonoyl-ACP

Assays are carried out in half-area, 96-well microtitre plates.Compounds are evaluated in 100 uL assay mixtures containing 100 mM MES,51 mM diethanolamine, 51 mM triethanolamine, pH6.5[MES=2-(N-morpholino)ethanesulfonic acid], 4% glycerol buffer, 100 mMNH₄Cl, 25 μM crotonoyl-ACP, 50 μM NADH, and 15 nM S. pneumoniae FabK.Inhibitors are typically varied over the range of 0.025-30 uM. Theconsumption of NADH is monitored for 30 minutes at 30° C. by followingthe change in absorbance at 340 nm. Initial velocities are estimatedfrom a linear fit of the progress curves. IC₅₀'s are estimated from afit of the initial velocities to a standard, 4-parameter model(Equation 1) and are typically reported as the mean±S.D. of duplicatedeterminations. Compounds of this invention in this assay have IC₅₀'sfrom about 60.0 micromolar to about 0.01 micromolar. The apparent K_(i)is calculated from Equation 2 assuming the inhibition is competitve withcrotonoyl-ACP.

Antimicrobial Activity Assay

Whole-cell antimicrobial activity is determined by broth microdilutionusing the National Committee for Clinical Laboratory Standards (NCCLS)recommended procedure, Document M7-A5, “Methods for DilutionSusceptibility Tests for Bacteria that Grow Aerobically”. The compoundis tested in serial two-fold dilutions ranging from 0.06 to 64 mcg/mL. Apanel of 12 strains are evaluated in the assay. This panel consists ofthe following laboratory strains: Enterococcus faecalis 29212,Staphylococcus aureus 29213, Staphylococcus aureus 43300, Moraxellacatarrhalis 49143, Haemophilus influenzae 49247, Streptococcuspneumoniae 49619, Staphylococcus epidermidis 1024939, Staphylococcusepidermidis 1024961, Escherichia coli AG100 (AcrAB⁺), Escherichia coliAG100A (AcrAB⁻), Pseudomonas aeruginosa K767 (MexAB⁺, OprM⁺),Pseudomonas aeruginosa K1119 (MexAB⁻, OprM⁻). The minimum inhibitoryconcentration (MIC) is determined as the lowest concentration ofcompound that inhibited visible growth. A spectrophotometer is used toassist in determining the MIC endpoint.

MIC assays may be performed using the microdilution method in a 96 wellformat. The assays may be performed in 96 well plates with a finalvolume of 100 μl cation-adjusted Mueller Hinton broth containing 2 foldserial dilutions of compounds ranging from 32 to 0.06 μg/ml. Bacterialgrowth may be measured at 600 nm using a Molecular Devices SpectraMax340PC spectrophotometer. MICs can then be determined by an absorbancethreshold algorithm and confirmed in some cases by inspecting the platesover a light box.

Minimum Bactericidal Concentration (MBC) may be determined by platingaliquots of MIC dilution series that did not show bacterial growth ontoPetri plates containing appropriate semi-solid growth media. The lowestcompound concentration that resulted in >99% killing of bacterial cells(relative to initial bacterial inocula in MIC test) is defined as theMBC.

Several strain panels may be used at various points in the compoundprogression scheme. The primary panel may include single prototypestrains of both community- and hospital-acquired pathogens fordetermining initial activities and spectra of activity. Secondary panelcompositions will depend on the results of the primary panels, and willinclude 10-20 strains of relevant species that will include communityacquired and antibiotic-resistant hospital acquired strains ofStaphylococcus aureus and coagulase negative Staphylococci together withother strains that are sensitive to the new compounds, and negativecontrol strains. The secondary panels will be used during optimizationof lead chemical series. Tertiary panels will include 100-200 clinicalstrains of S. aureus and coagulase negative Staphylococci together withother relevant strains as for the secondary panels. The tertiary panelswill be utilized during the compound candidate selection stage andpreclinical studies to generate bacterial population efficacy parameterssuch as MIC₅₀ and MIC₉₀.

Franciscella Tularensis In Vitro Efficacy Studies

Routine MIC testing of F. tularensis may be undertaken on compounds thathave demonstrated enzymatic activity inhibition against the F.tularensis FabI protein. The MIC testing of F. tularensis may beoutsourced to a facility with BL3 capabilities, and with experience inhandling F. tularensis cultures in the laboratory. The studies may beundertaken with the recommended methods for antimicrobial susceptibilitytesting of F. tularensis.

Helicobacter pylori In Vitro Efficacy Studies

Routine MIC testing of H. pylori may be undertaken on compounds thathave demonstrated enzymatic activity inhibition against the H. pyloriFabI protein. The studies may be undertaken with the recommended methodsfor antimicrobial susceptibility testing of H. pylori.

Cytotoxicity Assays

Cytotoxicity of the new compounds may be evaluated by the Alamar Blueassay according the manufacturers instructions. Human cell lines (e.g.Jurkat) grown in 96 well plates may be exposed to serial dilutions ofthe tested compounds. After adding Alamar Blue, cell viability may bedetermined by measuring the absorbance of the reduced and oxidized formsof Alamar Blue at 570 nm and 600 nm. Cytotoxicity may be reported asLD₅₀, the concentration that causes a 50% reduction in cell viability.

Dosages

The dosage of any compositions of the present invention will varydepending on the symptoms, age and body weight of the patient, thenature and severity of the disorder to be treated or prevented, theroute of administration, and the form of the subject composition. Any ofthe subject formulations may be administered in a single dose or individed doses. Dosages for the compositions of the present invention maybe readily determined by techniques known to those of skill in the artor as taught herein.

In certain embodiments, the dosage of the subject compounds willgenerally be in the range of about 0.01 ng to about 10 g per kg bodyweight, specifically in the range of about 1 ng to about 0.1 g per kg,and more specifically in the range of about 100 ng to about mg per kg.

An effective dose or amount, and any possible affects on the timing ofadministration of the formulation, may need to be identified for anyparticular composition of the present invention. This may beaccomplished by routine experiment as described herein, using one ormore groups of animals (preferably at least 5 animals per group), or inhuman trials if appropriate. The effectiveness of any subjectcomposition and method of treatment or prevention may be assessed byadministering the composition and assessing the effect of theadministration by measuring one or more applicable indices, andcomparing the post-treatment values of these indices to the values ofthe same indices prior to treatment.

The precise time of administration and amount of any particular subjectcomposition that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a subject composition, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, and the like. The guidelines presented hereinmay be used to optimize the treatment, e.g., determining the optimumtime and/or amount of administration, which will require no more thanroutine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during the treatment period. Treatment, includingcomposition, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters. Adjustments to the amount(s) of subjectcomposition administered and possibly to the time of administration maybe made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage forany individual agent contained in the compositions (e.g., the FabIinhibitor) because the onset and duration of effect of the differentagents may be complimentary.

Toxicity and therapeutic efficacy of subject compositions may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ and the ED₅₀.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany subject composition lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For compositions ofthe present invention, the therapeutically effective dose may beestimated initially from cell culture assays.

Combinations

Compositions are also contemplated herein that include one or more ofthe disclosed antibacterial compounds with a second component. Secondcomponents in such antibacterial compositions of the present inventionare usually an antibiotic agent other than a FabI inhibitor. Additionalcomponents may also be present, including other FabI inhibitors orantibiotic agents.

Non-limiting examples of antibiotic agents that may be used in theantibacterial compositions of the present invention includecephalosporins, quinolones and fluoroquinolones, penicillins,penicillins and beta lactamase inhibitors, carbepenems, monobactams,macrolides and lincosamines, glycopeptides, rifampin, oxazolidonones,tetracyclines, aminoglycosides, streptogramins, sulfonamides, andothers. Each family comprises many members.

Cephalosporins are further categorized by generation. Non-limitingexamples of cephalosporins by generation include the following. Examplesof cephalosporins I generation include Cefadroxil, Cefazolin,Cephalexin, Cephalothin, Cephapirin, and Cephradine. Examples ofcephalosporins II generation include Cefaclor, Cefamandol, Cefonicid,Cefotetan, Cefoxitin, Cefprozil, Ceftmetazole, Cefuroxime, Cefuroximeaxetil, and Loracarbef. Examples of cephalosporins III generationinclude Cefdinir, Ceftibuten, Cefditoren, Cefetamet, Cefpodoxime,Cefprozil, Cefuroxime (axetil), Cefuroxime (sodium), Cefoperazone,Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime. Ceftizoxime,and Ceftriaxone. Examples of cephalosporins IV generation includeCefepime.

Non-limiting examples of quinolones and fluoroquinolones includeCinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin,Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin,Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, andPerfloxacin.

Non-limiting examples of penicillins include Amoxicillin, Ampicillin,Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, andTicarcillin.

Non-limiting examples of penicillins and beta lactamase inhibitorsinclude Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam,Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin,Penicillin G (Benzathine, Potassium, Procaine), Penicillin V,Piperacillin+Tazobactam, Ticarcillin+Clavulanic Acid, and Nafcillin.

Non-limiting examples of carbepenems include Imipenem-Cilastatin andMeropenem.

A non-limiting example of a monobactam includes Aztreonam. Non-limitingexamples of macrolides and lincosamines include Azithromycin,Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin,and Troleandomycin. Non-limiting examples of glycopeptides includeTeicoplanin and Vancomycin. Non-limiting examples of rifampins includeRifabutin, Rifampin, and Rifapentine. A non-limiting example ofoxazolidonones includes Linezolid. Non-limiting examples oftetracyclines include Demeclocycline, Doxycycline, Methacycline,Minocycline, Oxytetracycline, Tetracycline, and Chlortetracycline.

Non-limiting examples of aminoglycosides include Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, andParomomycin. A non-limiting example of streptogramins includesQuinopristin+Dalfopristin.

Non-limiting examples of sulfonamides include Mafenide, SilverSulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole,Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, andSulfamethizole.

Non-limiting examples of other antibiotic agents include Bacitracin,Chloramphenicol, Colistemetate, Fosfomycin, Isoniazid, Methenamine,Metronidazol, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin,Polymyxin B, Spectinomycin, Trimethoprim, Colistin, Cycloserine,Capreomycin, Pyrazinamide, Para-aminosalicyclic acid, and Erythromycinethylsuccinate+sulfisoxazole.

Formulations

The antibacterial compositions of the present invention may beadministered by various means, depending on their intended use, as iswell known in the art. For example, if compositions of the presentinvention are to be administered orally, they may be formulated astablets, capsules, granules, powders or syrups. Alternatively,formulations of the present invention may be administered parenterallyas injections (intravenous, intramuscular or subcutaneous), dropinfusion preparations or suppositories. For application by theophthalmic mucous membrane route, compositions of the present inventionmay be formulated as eyedrops or eye ointments. These formulations maybe prepared by conventional means, and, if desired, the compositions maybe mixed with any conventional additive, such as an excipient, a binder,a disintegrating agent, a lubricant, a corrigent, a solubilizing agent,a suspension aid, an emulsifying agent or a coating agent.

In formulations of the subject invention, wetting agents, emulsifiersand lubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants may bepresent in the formulated agents.

Subject compositions may be suitable for oral, nasal, topical (includingbuccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of composition that may be combined with a carriermaterial to produce a single dose vary depending upon the subject beingtreated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringinginto association compositions of the present invention with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation agents with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition thereof as an active ingredient. Compositions of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject composition ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

Formulations and compositions may include micronized crystals of thedisclosed compounds. Micronization may be performed on crystals of thecompounds alone, or on a mixture of crystals and a part or whole ofpharmaceutical excipients or carriers. Mean particle size of micronizedcrystals of a disclosed compound may be for example about 5 to about 200microns, or about 10 to about 110 microns.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the subject composition, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, cyclodextrins and mixturesthereof.

Suspensions, in addition to the subject composition, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and release theactive agent. Formulations which are suitable for vaginal administrationalso include pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for transdermal administration of a subject compositionincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. The active component may be mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to asubject composition, excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays may additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Compositions and compounds of the present invention may alternatively beadministered by aerosol. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containing thecompound. A non-aqueous (e.g., fluorocarbon propellant) suspension couldbe used. Sonic nebulizers may be used because they minimize exposing theagent to shear, which may result in degradation of the compoundscontained in the subject compositions.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of a subject composition together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularsubject composition, but typically include non-ionic surfactants(Tweens, Pluronics, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a subject composition in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate and cyclodextrins. Proper fluidity may be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In certain embodiments, the subject compounds may be formulated as atablet, pill capsule or other appropriate ingestible formulation(collectively hereinafter “tablet”), to provide a therapeutic dose in 10tablets or fewer. In another example, a therapeutic dose is provided in50, 40, 30, 20, 15, 10, 5 or 3 tablets.

In a certain embodiment, the antibacterial agent is formulated for oraladministration as a tablet or an aqueous solution or suspension. Inanother embodiment of the tablet form of the antibacterial agent, thetablets are formulated such that the amount of antibacterial agent (orantibacterial agents) provided in 20 tablets, if taken together, wouldprovide a dose of at least the median effective dose (ED₅₀), e.g., thedose at which at least 50% of individuals exhibited the quantal effectof inhibition of bacterial cell growth or protection (e.g., astatistically significant reduction in infection). In a furtherembodiment, the tablets are formulated such that the total amount ofantibacterial agent (or antibacterial agents) provided in 10, 5, 2 or 1tablets would provide at least an ED₅₀ dose to a patient (human ornon-human mammal). In other embodiments, the amount of antibacterialagent (or antibacterial agents) provided in 20, 10, 5 or 2 tablets takenin a 24 hour time period would provide a dosage regimen providing, onaverage, a mean plasma level of the antibacterial agent(s) of at leastthe ED₅₀ concentration (the concentration for 50% of maximal effect of,e.g., inhibiting bacterial cell growth). In other embodiments less than100 times, 10 times, or 5 times the ED50 is provided. In otherembodiments, a single dose of tablets (1-20 tablets) provides about 0.25mg to 1250 mg of an antibacterial agent(s).

Likewise, the antibacterial agents can be formulated for parenteraladministration, as for example, for subcutaneous, intramuscular orintravenous injection, e.g., the antibacterial agent can be provided ina sterile solution or suspension (collectively hereinafter “injectablesolution”). The injectable solution is formulated such that the amountof antibacterial agent (or antibacterial agents) provided in a 200 ccbolus injection would provide a dose of at least the median effectivedose, or less than 100 times the ED₅₀, or less than 10 or times theED₅₀. The injectable solution may be formulated such that the totalamount of antibacterial agent (or antibacterial agents) provided in 100,50, 25, 10, 5, 2.5, or 1 cc injections would provide an ED₅₀ dose to apatient, or less than 100 times the ED₅₀, or less than 10 or 5 times theED₅₀. In other embodiments, the amount of antibacterial agent (orantibacterial agents) provided in a total volume of 100 cc, 50, 25, 5 or2 cc to be injected at least twice in a 24 hour time period wouldprovide a dosage regimen providing, on average, a mean plasma level ofthe antibacterial agent(s) of at least the ED₅₀ concentration, or lessthan 100 times the ED₅₀, or less than 10 or 5 times the ED₅₀. In otherembodiments, a single dose injection provides about 0.25 mg to 1250 mgof antibacterial agent.

Efficacy of Treatment

The efficacy of treatment with the subject compositions may bedetermined in a number of fashions known to those of skill in the art.

In one exemplary method, the median survival rate of the bacteria orbacteria median survival time or life span for treatment with a subjectcomposition may be compared to other forms of treatment with theparticular FabI inhibitor, or with other antibiotic agents. The decreasein median bacteria survival rate or time or life span for treatment witha subject composition as compared to treatment with another method maybe 10, 25, 50, 75, 100, 150, 200, 300, 400% even more. The period oftime for observing any such decrease may be about 3, 5, 10, 15, 390, 60or 90 or more days. The comparison may be made against treatment withthe particular FabI inhibitor contained in the subject composition, orwith other antibiotic agents, or administration of the same or differentagents by a different method, or administration as part of a differentdrug delivery device than a subject composition. The comparison may bemade against the same or a different effective dosage of the variousagents. The different regiments compared may use measurements ofbacterial levels to assess efficacy.

Alternatively, a comparison of the different treatment regimensdescribed above may be based on the effectiveness of the treatment,using standard indicies for bacterial infections known to those of skillin the art. One method of treatment may be 10%, 20%, 30%, 50%, 75%,100%, 150%, 200%, 300% more effective, than another method.

Alternatively, the different treatment regimens may be analyzed bycomparing the therapeutic index for each of them, with treatment with asubject composition as compared to another regimen having a therapeuticindex two, three, five or seven times that of, or even one, two, threeor more orders of magnitude greater than, treatment with another methodusing the same or different FabI inhibitor.

As a non-limiting example, to determine if compounds are bactericidal orbacteriostatic at relevant concentrations, and to examine the kineticsof bacterial killing the following experiment may be performed with S.aureus, S. epidermidis and appropriate control strains and antibiotics.To fresh logarithmic cultures at 10⁷ viable cells/ml, compound may beadded to reach concentrations of X1, X2 or X4 the MIC. Control cultureswill receive no compound. At 1 hour intervals, aliquots will be dilutedand plated for determining viable counts. Plots of viable cells vs. timefor up to 24 hours will reveal bactericidal/bacteriostatic properties ofthe compounds, and also show the kill kinetics. These experiments areimportant to determine whether these inhibitors have time-dependent orconcentration-dependent effects, and will be used to help setappropriate dosages in vivo in combination with pharmacokinetic andpharmacodynamic measurements.

In the practice of the instant methods, the antibacterial compositionsof the present invention inhibit bacterial FabI with a K_(i) of 5 μM orless, 1 μM or less, 100 nM or less, 10 nM or less or even 1 nM or less.In treatment of humans or other animals, the subject method may employFabI inhibitors which are selective for the bacterial enzyme relative tothe host animals' enoyl CoA hydratase, e.g., the K_(i) for inhibition ofthe bacterial enzyme is at least one order, two orders, three orders, oreven four or more orders of magnitude less than the K_(i) for inhibitionof enoyl CoA hydratase from the human (or other animal). That is, thepractice of the subject method in vivo in animals utilizes FabIinhibitors with therapeutic indexes of at least 10, 100 or 1000.

Similarly, in the practice of the instant method, the antibacterialcompounds of the present invention inhibit FabI with an IC₅₀ of 30 μM orless, 10 μM or less, 100 nM or less, or even 10 nM or less. In treatmentof humans or other animals, the subject method may employ FabIinhibitors which are selective for the bacterial enzyme relative to thehost animals' enoyl CoA hydratase, e.g., the IC₅₀ for inhibition of thebacterial enzyme is at least one order, two orders, three orders, oreven four orders of magnitude less than the IC₅₀ for inhibition of enoylCoA hydratase from the human (or other animal). That is, in preferredembodiments, the practice of the subject method in vivo in animalsutilizes FabI inhibitors with therapeutic indexes of at least 10, 100 or1000.

Alternatively, bacterial inhibition by an antibacterial compound of thepresent invention may also be characterized in terms of the minimuminhibitory concentration (MIC), which is the highest concentration ofcompound required to achieve complete inhibition of bacterial cellgrowth. Such values are well known to those in the art as representativeof the effectiveness of a particular antibacterial agent against aparticular organism or group of organisms. In the practice of theinstant methods, the antibacterial compositions of the present inventioninhibit bacterial growth with MIC values of about 32 μg/mL, less thanabout 16 μg/mL, less than about 8 μg/mL, less than about 4 μg/mL, lessthan about 2 μg/mL, less than about 1 μg/mL, less than about 0.5 μg/mL,less than about 0.25 μg/mL, or even less than about 0.125 Ag/mL. Thevalue of MIC900, defined as the concentration of a compound required toinhibit the growth of 90% of bacterial strains within a given bacterialstrain population, can also be used. In certain embodiments, thecompounds of the present invention are selected for use based, interalia, on having MIC90 values of less than about 32 μg/mL, less thanabout 16 μg/mL, less than about 8 μg/mL, less than about 4 μg/mL, lessthan about 2 μg/mL, less than about 1 μg/mL, less than about 0.5 μg/mL,less than about 0.25 μg/mL, or even less than about 0.125 μg/mL.

In other embodiments, the subject compounds are selected for use inanimals, or animal cell/tissue culture based at least in part on havingLD₅₀'s at least one order, or two orders, or three orders, or even fourorders or more of magnitude greater than the ED₅₀. That is, in certainembodiments where the subject compounds are to be administered to ananimal, a suitable therapeutic index is preferably greater than 10, 100,1000 or even 10,000.

Kits

This invention also provides kits for conveniently and effectivelyimplementing the methods of this invention. Such kits comprise anysubject composition, and a means for facilitating compliance withmethods of this invention. Such kits provide a convenient and effectivemeans for assuring that the subject to be treated takes the appropriateactive in the correct dosage in the correct manner. The compliance meansof such kits includes any means which facilitates administering theactives according to a method of this invention. Such compliance meansinclude instructions, packaging, and dispensing means, and combinationsthereof. Kit components may be packaged for either manual or partiallyor wholly automated practice of the foregoing methods. In otherembodiments involving kits, this invention contemplates a kit includingcompositions of the present invention, and optionally instructions fortheir use.

The examples which follow are intended in no way to limit the scope ofthis invention but are provided to illustrate how to prepare and usecompounds of the present invention. Many other embodiments of thisinvention will be apparent to one skilled in the art.

EXAMPLES General

FIG. 1 shows a 96-well plate after the addition of acid, after thecooling down of the plate, and after evaporation of solvent usingScheme 1. Red wells were solids that precipitated out of solution uponthe addition of the acid to the Compound A solution. Blue wells weresolids that precipitated out of solution when the plate had reached roomtemperature. Purple wells had solids after removal of the solvent. Wellswith no color are wells that did not produce solids. The black “X” inthe DCM/sulfuric is a well in which the solid disappeared, possible dueto this solid being deliquescent. The 11^(th) “blank” well was chargedwith(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide(compound A) and used as a control to monitor the crystallization offree base in the different solvents.

Determination of crystallinity was performed on solid-containing wellsby dispersing the solids in mineral oil and using a polarized lightmicroscope at 100× and 400×. Samples which contained particles withdistinct birefringence and extinction positions were identified ascrystalline. Those which had some particles which exhibitedbirefringence and extinction positions but also had a significant amountof particles which were not observed to reflect light were described aspartially crystalline. Amorphous solids were those which did not havesignificant amount of particles that reflected polarized light whiledeliquescent material either became liquid-like or softened duringanalysis. Counter-ions which were found to exhibit the crystalline topartially crystalline material included maleic. TCA, TFA, HCl,phosphoric acid, benzenesulfonic, methansulfonic, HBr, and sulfuric. Allsolvents produced crystalline material with THF, AcOH, DCM, and DMAproducing at least 3 crystalline solids.

Example 1(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidehydrochloride

Using method IIA, the vial containing the starting compound A, HCl andDCM solvent, upon cooling, yielded the title compound as a white solid.XRPD analysis indicated a crystalline solid distinct from the free base(compound A). The XRPD spectra is as shown in FIG. 3. High intensitypeaks include those occurring at 6.75, 8.25, 12.5, 16, 17, 17.75, 18,20.25, 20.5, 23, 24.5, 27.5, 29, 31, and 32 2θ. The DSC of the solid HCLsalt showed two thermal events, as depicted in FIG. 3.

Example 2(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidetrifluoroacetate

Using method IIA, the vial containing compound A, trifluoroacetic acidand acetic acid solvent, upon cooling, yielded the title compound as awhite solid. XRPD analysis indicated a crystalline solid distinct fromthe free base.

Example 3(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidemethane sulfonate

Compound A, methane sulfonic acid and THF were processed as described inmethod IIB (Table 1) above. A solid crystallized during the coolingphase which was isolated as the title compound. The product showed aunique diffraction (XRPD) as compared to the free base as shown in FIG.4. High intensity peaks include those occurring at 7, 14, 15.5, 20, 21,23, 28, 35.5 and 42.5 2θ. Two thermal events were seen (DSC)

In a similar manner from DCM upon concentration (about half volumereduction), crystallization occurred to yield the title compound: uniqueXRPD; mp 187° C.; (DSC); ¹H-NMR supports an assignment with the ratiobetween compound A and counter-ion being 1:1; the material also showedno retained solvent by weight loss in the TGA.

Example 4(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidebenzene sulfonate

Compound A, benzene sulfonic acid and THF were processed as described inmethod IIB (Table 1). A solid crystallized during the cooling phasewhich was isolated as the title compound. The product showed a uniquediffraction (XRPD) as compared to the free base as shown in FIG. 5;(high intensity peaks include those occurring at 6, 12, 23.5 and 31 2θ);mp 172° C. (DSC); the ¹H-NMR supported a ratio between the free base andcounter-ion of 1:1.

In a similar manner from DCM upon concentration (about half volumereduction), crystallization occurred to yield the title compound: uniqueXRPD as shown in FIG. 6 (high intensity peaks include those occurring at5.5, 6, 13, 15.5, 18 and 19 2θ); mp 168° C.; (DSC), ¹H-NMR supports anassignment with the ratio between free base and counter-ion being 1:1.

Example 5(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidep-toluene sulfonate

Using method IIB, compound A, p-toluene sulfonic acid and THF assolvent, a solid crystallized during the cooling phase which wasisolated as the title compound. The product showed a unique diffraction(XRPD) as compared to the free base as shown in FIG. 7A (high intensitypeaks include those occurring at 6.5, 11.5, 12.25, 13.5, 14.5, 14.25,15.5, 16, 18.5, 19, 26, and 27 2θ); mp 180.5° C. (DSC); ¹H-NMR supportsa ratio between compound a and counter-ion of 1:1; no weight loss inTGA. The DCS of this salt from THF is depicted in FIG. 7B.

In a similar manner from DCM upon concentration (about half volumereduction), crystallization occurred to yield the title compound: uniqueXRPD; three thermal events in DSC, ¹H-NMR supports an assignment withthe ratio between compound A and counter-ion of 1:1.

Example 6(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidesulfate

Compound A, sulfuric acid and THF were processed as described in methodIIB (Table 1) to yield the title compound as a crystalline solid. Theproduct produced a unique XRPD pattern (FIG. 8) compared to thefree-base as well as a different (lower) thermal event in the DSC. Highintensity peaks include those occurring at 4.5, 10, 14.75, 14, 22, 24.5,and 26 2θ.

Example 7(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidemethane sulfonate

Using general method III, described above, the title salt was preparedin a yield of 84.4% with analytical data consistent with thosepreviously described for example 3: elemental analysis calculated forC₂₃H₂₆N₃O₆.CH₃SO₃H as a monohydrate: C, 56.43; H, 5.56; N, 8.58. Found:C, 56.10. H, 5.38; N, 8.46.

A portion of this material was subjected to ball milling as describedabove; the XRPD shows crystalline material as depicted in FIG. 9A; DSC(FIG. 9B) shows one major thermal event and a decomposition exotherm.

Example 8(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidep-toluene sulfonate

Using general method III, described above, the title salt was preparedin a yield of 77.8% with analytical data consistent with thosepreviously described for example 5: Analysis calculated for C₂₃H₂₆N₃O₆.pCH₃PhSOH: C, 63.60; H, 5.34; N, 7.67. Found: C, 63.31; H, 5.44; N,7.56.

A portion of this material was subjected to ball milling as describedabove: XRPD shows crystalline material as depicted in FIG. 10A; DSC(FIG. 10 B) shows one thermal event.

Example 9(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidesulfate

Using method III, described above, the title salt was prepared in ayield of 84.4% with analytical data consistent with those previouslydescribed for example 6: Analysis calculated for C₂₁H₂₆N₃O₆.H₂SO₄: C,55.80; H, 4.90. N, 8.87. Found: C, 56.00. H, 5.46; N, 7.87. The DSC isdepicted in FIG. 11.

A portion of this material was subjected to ball milling as describedabove: XRPD shows amorphous material is obtained.

Example 10

(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidep-toluene sulfonate Monohydrate form: Large-scale process (Scheme II,vida supra)

A. N,3-dimethylbenzofuran-2-carboxamide

A mixture of 3-methylbenzofuran-2-carboxylic acid (1.0 kg, 5.676 mol) inmethylene chloride (5.8 L) and DMF (5 mL) was chilled to ˜2° C. Asolution of oxalyl chloride (864 g, 6.81 mol) was added to the reactionmixture keeping the temperature below 10° C., over a period of 2 hrs. Avigorous evolution of a gas was observed. The reaction mixture wasstirred overnight under nitrogen at room temperature and then refluxedfor 3 hrs. All of the solids were dissolved to give a brown colorsolution. HPLC analysis of an aliquot indicated that the reaction wascomplete. The mixture was concentrated on a rotary evaporator and theresidual oxalyl chloride was chased with DCM (2 L). The solid productwas re-dissolved in DCM (6 L) in a round bottom flask (10 L) and chilledto ˜−5° C. A solution of methyl amine (40% in water, 1.7 L, 3.5 eq,19.86 mol) was carefully added to the acid chloride solution whilekeeping the temperature below 8° C. then stirred overnight at roomtemperature. Analysis of an aliquot indicated that the reaction wascomplete. Water (8.0 L) was added to the reaction mixture, the layerswere separated, the aqueous layer was back extracted with DCM (2×3 L),the combined organic layer was washed with water (4 L) and dried overNa₂SO₄. The organic layer was filtered, the filtrate was concentratedand traces of DCM were co evaporated with heptanes (2 L). The heptanesslurry was filtered and the cake was washed with heptanes (2×2 L). Allthe brown color was removed in the heptane filtrate. The solid productwas dried under high vacuum overnight to a constant weight. Yield of thetitle compound was 1.015 kg (94.5%. ¹H NMR {CDCl₃}: δ (ppm) 8.45 (s,1H), 7.61 (d, 1H), 7.41 (m, 2H), 7.30 (m, 1H), 3.03 (d, 2H), 2.63 (s,3H). HPLC: 99.0 area % (R_(T)=7.91 min)

B. N-methyl-1-(3-methylbenzofuran-2-yl)methanamine

To a solution of THF (3.4 L) in a 22 L round bottom flask were carefullyadded lithium aluminum hydride pellets (261 g, 6.871 mol, 1.3 eq) andthe mixture was stirred over night under a nitrogen atmosphere. Most ofthe pellets dissolved and a gray slurry formed. The mixture was chilledto 5° C. and a solution of N,3-dimethylbenzofuran-2-carboxamide (1.0 kg,5.285 mol) in THF (7.7 L) was added carefully while maintaining theinternal temperature below 10° C. The addition took ˜1.5 hrs. Then thereaction mixture was carefully refluxed for 7 hrs and HPLC analysis atthis stage indicated 85.9% of product, 13.9% of starting material and1.2% of a side product. At this stage the heating was stopped, theheating mantle was replaced with a cooling bath and the mixture wascooled to −20° C. The reaction mixture was carefully quenched with water(330 mL) keeping the temperature below 0° C., followed by addition of anaqueous solution of 2 N NaOH (400 mL) and the resulting mixture wasallowed to stand over night. A lot of greasy residue settled to thebottom the upper layer was turbid and was very difficult to filter. TheTHF layer was concentrated under vacuum and the residue was dissolved inethyl acetate (4 L) and filtered (very slow filtration). The filter cakewas washed with EtOAc (4×4 L) and the combined filtrate was concentratedon a rotary evaporator to obtain ˜1.119 kg of crude oil. Totalfiltration time is about 2 days.

Similarly another 1.2 kg reaction (313 g of LAH) was completed and afterwork-up 1.5 kg of oil was isolated.

Purification of the combined crude oil by acid base work-up and silicagel chromatography yielded the title compound as a yellow oil (71.3%).¹H NMR {CDCl₃}: δ (ppm) 7.47 (d, 1H), 7.39 (d, 1H), 7.22 (m, 2H), 3.82(s, 2H), 2.43 (s, 3H), 2.24 (s, 3H) HPLC: 98.3 area % (R_(T)=5.59 min)

C. (E)-tert-butyl3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylate

A 22-L three-necked round-bottomed flask with a mechanical stirrer, athermocouple, a reflux condenser and a nitrogen inlet was flushed withN₂. The flask was charged with acetonitrile (8 L, 5 v),dimethylformamide (93.2 L, 2 v) and6-bromo-3,4-dihydro-1,8-naphthyridin-2(1H)-one bromide (1.6 kg, 7.05mol, 1 eq.). To the suspension were added diisopropylethylamine (1.33kg, 10.57 mol, 1.5 eq.) and tert-butylacrylate (1.33 kg, 10.57 mol, 1.5eq.). The suspension was then purged with a slow stream of N₂ for 10min. Trio-tolylphosphine (0.214 kg, 0.705 mol, 0.1 eq) and palladiumdiacetate (0.079 kg, 0.352 mol, 0.05 eq) were added to the reactionmixture. The reaction mixture was heated to 75-80° C. and stirred at75-80° C. for 20 h. The reaction mixture was cooled to 20-25° C.,diluted with water (10 v) and stirred for one hour. The solid wascollected by vacuum filtration using a Buchnner funnel with a clothfilter and 20-L glass receiver flask. The solids were then washed withwater (3×1 v) followed by heptane (2×1 v). The light brown damp solidwas dried under vacuum at 30-35° C. with a slow N₂ bleed for 48 h to aconstant weight to give 2.11 kg of light brown solid. This solid wasthen suspended DCM/MeOH (10 v/1 v) under N₂ in two 22-L three neckedRBFs with a mechanical stirrer, a thermocouple and a reflux condenser.To each of these RBFs charcoal (100 g, ALDRICH Activated Darco G 60-100mesh) was added and the mixture allowed to stir at 20-25° C. for 20 h.The suspensions were filter through a pad of celite (500 g); silica gel(500 g) using a cintered glass funnel and a 20-L glass filter flask. Thesolid cake was rinsed with DCM (3×1 v). The combined filtrate wasconcentrated under vacuum ˜30-35° C. and the residue was diluted withheptane/ethyl acetate (3 v/3 v) and further concentrated to asuspension. The solid product was collected under vacuum by filtrationusing a Buchnner funnel with a cloth filter. The solids were rinsed withheptane (3×1.25 v) and the damp solid was dried under vacuum at 30-35°C. with a slow N₂ bleed for 90 h to a constant weight to give 1.58 kg(82%) of the desired product as a yellow solid.

¹H NMR {DMSO (d₆)}: δ (ppm) 10.63 (br, 1H, NH), 8.36 (s, 1H, CH), 8.01(s, 1H, CH), 7.52 (d, J=15.6 Hz, 1H, CH), 6.51 (d, J=15.6, Hz, 1H, CH),2.93 (t, J=7.9 Hz, 2H, CH₂), 2.56 (t, J=7.9 Hz, 2H, CH₂), 1.51 (s, 9H,C(CH₃)₃)

¹³C NMR {DMSO (ds)}:}: δ (ppm) 171.4 (C), 165.9 (C), 153.3, 147.8,140.7, 134.2, 125.1, 119.5, 119.4, 80.3 (C), 30.4 (CH₂), 28.3 (CH₃) &23.7 (CH₂).

HPLC: 98.0 area % (R_(T)=11.52 min)

D. (E)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylic acid

A 22-L three-necked round-bottomed flask with a mechanical stirrer, athermocouple, a reflux condenser and a nitrogen inlet was flushed withN₂ then charged with glacial acetic acid (12.5 L, 7 v) and(E)-tert-butyl3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylate (1.58 kg,5.76 mol, 1.0 eq.). To the yellow suspension, HBr (2.91 kg, 17.28 mol, 3eq., 48% aq.) was added. The resulting suspension was stirred at 20-25°C. Due to difficulty of stirring, acetic acid (7.5 L) was added. Afterstirring for 2.5 h, ˜1 mL of yellow suspension was taken out, filtered,washed with ethyl acetate and dried. The dry solid dissolved in CDCl₃and the progress of the reaction was monitored by ¹H NMR. The resultindicated >90% reaction completion. After another 16 hours of stirringat the same temperature, the ¹H NMR analysis indicated the reaction wascomplete. The solid was collected by filtration using a Buchnner funnelwith a cloth filter (WHATMAN PAPER Cat#1821915) and 20-L glass receiverunder vacuum. The solids were washed with ethyl acetate (2×2 v). Thedamp solid was transferred into a 20-L plastic bucket containing ethylacetate (12 L, 8 v) and agitated with a mechanical stirre. The resultingsuspension was stirred at 20-25° C. for 24 h. Solid was collected byfiltration using a Buchnner funnel with a cloth filter and 20-L glassreceiver under vacuum. The solids were washed with ethyl acetate (3×2 V)and the damp solid was dried under vacuum at 30-35° C. with a slow N₂bleed for 60 h to a constant weight to give 1.60 kg of the HBr salt ofthe desired product as a yellow solid.

¹H NMR {DMSO (d₆)}: δ (ppm) 10.4-10.8 (br, 2H, HBr, NH), 8.36 (s, 1H,CH), 8.01 (s, 1H, CH), 7.55 (d, J=16.2 Hz, 1H, CH), 6.51 (d, J=16.2, Hz,1H, CH), 2.93 (t, J=7.2 Hz, 2H, CH₂), 2.56 (t, J=7.2 Hz, 2H, CH₂), 1.51(s, 9H, C(CH₃)₃)

¹³C NMR {DMSO (d₆)}:}: δ (ppm) 171.5 (C), 167.9 (C), 153.0, 147.1,140.1, 134.8, 125.3, 120.1, 119.2, 30.4 (CH₂), & 23.8 (CH₂).

HPLC: 99.6 area % (R_(T)=6.70 min)

E.(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide

A 22-L three-necked round-bottomed flask with a mechanical stirrer, athermocouple, a reflux condenser and a nitrogen inlet was flushed withN₂. The flask was charged with DMF (7.5 L, 5 v) and(E)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylic acid (1.49kg, 4.98 mol, 1.0 eq.). To the yellow suspension, HOBT (0.74 kg, 5.48mol, 1.1 eq.), EDCI (1.15 kg, 5.98 mol, 1.2 eq) andN-methyl-1-(3-methylbenzofuran-2-yl)methanamine (0.96 kg, 5.48 mol, 1.1eq) were added. During the addition an exotherm was observed and thetemperature increased from 20.3° C. to 36.4° C. To the resulting thicksuspension was added DIPEA (1.93 kg, 14.94 mol, 3 eq.) and an exothermwas observed to 41.9° C. During the addition the suspension changed to aclear brown solution. The resulting solution was stirred at 38-42° C.After about 45 min a new solid started to form. After stirring for 1.5h, ˜1 mL of suspension was taken, the progress of the reaction wasmonitored by HPLC and the results indicated >95% reaction completion.After another 16 hours of stirring at the same temperature, ¹H NMR andHPLC analysis indicated the reaction was complete. Half of the reactionmixture was transferred to another 22-L three-necked round-bottomedflask containing a mechanical stirrer, a thermocouple, a refluxcondenser and a nitrogen inlet. The contents of both flasks were allowedto cool to 20-25° C. and each reaction mixture was diluted with H₂O (8L, 5.3 v) over 15 min. Both reaction mixtures showed an exotherm duringthe water addition to 30-35° C. The resulting suspensions were allowedto stir at 20-25° C. for 3.5 h. Solid was collected by filtration usinga Buchnner funnel with a cloth filter and 20-L glass receiver undervacuum. The solids were washed with water (4×1.4 v), heptane (1×1.4 v)and ethyl acetate (3×1 v). The damp solid was dried under vacuum at30-35° C. with a slow N₂ bleed for 120 h to a constant weight to give1.67 kg of the title compound as yellow solid. The material was analyzedindicating 98.48 area % HPLC purity with 146 ppm residual Pd.

A 22-L three-necked round-bottomed flask with a mechanical stirrer, athermocouple, a reflux condenser and a nitrogen inlet was flushed withN₂. The flask was charged with DMF (7 L, 4 v) and a potion of the crudeproduct from above (1.65 kg, 4.98 mol, 1.0 eq.). To the resulting thicksuspension was added DIPEA (1 L, 0.75 v.). The suspension was heated to52-57° C. and stirred at the same temperature. After 20 hours ofstirring at 52-57° C. the suspension was allowed to cool to 20-25° C.Solid was collected by filtration using a Buchnner funnel with a clothfilter and 20-L glass receiver under vacuum. The solids were washed withDMF (2×0.7 v), H₂O (3×2 v) and methanol (2×2 v). The damp solid wasdried under vacuum at 30-35° C. with a slow N₂ bleed for 110 h to aconstant weight to give 1.56 kg of the desired product as a yellowsolid. The material was analyzed and the results indicated 99 area %HPLC purity with 20 ppm Pd. A 0.75 kg of this material was used for thesalt formation and the remaining 0.813 kg was subjected to the procedureas described above beginning with suspension in DMF. A 0.78 kg portionof desired product was isolated as solid in 95% yield with 99.0 area %HPLC purity with 14 ppm of residual Pd. This material was used forsubsequent milling (Micron technologies).

¹H NMR {DMSO (d₆)}: δ (ppm) 10.69 (br, H, NH), 8.38 (two s, 1H, CH),8.09 (two s, 1H, CH), 7.48-7.58 (m, 3.4H), 7.19-7.31 (m, 2.5H), 5.01 (s,0.8H), 4.80 (s, 1.2H), 3.37 (s, 0.4H), 3.20 (s, 1.6H) 2.89-2.94 (m, 2H),2.50-2.94 (m, 5H, CH₂, CH₃), 2.27 (s, 3H, CH₃)

¹³C NMR {DMSO (d₆)}:}: δ (ppm) 171.5 (C), 165.9 (C), 154.0, 152.9,149.8, 147.7, 139.2, 138.8, 134.2, 126.1, 125.0, 124.8, 122.9, 120.0,119.5, 118.0, 111.3, 42.4 (CH₂), 35.4 (CH₃), 30.5 (CH₂), 23.8 (CH₂) &8.0 (CH₃)

DSC: 245.4° C.

TGA: 0.2652@ 240° C.

HPLC: 99.7 area % (R_(T)=11.52 min)

LC-MS: 376 amu (MW. 376.17)

KF: 0.23%

Pd: 16 ppm

Heavy Metals: <20 ppm

Residue on Ignition: 0.16%

C₂₁H₂₂N₃O₃: Calcd. C, 70.38%; H, 5.64%; N, 11.19%

Found C, 70.08%; H, 5.57%4; N, 11.17%

F.(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideTosylate Monohydrate

A 22-L three-necked round-bottomed flask with a mechanical stirrer, athermocouple, a reflux condenser and a nitrogen inlet was flushed withN₂. The flask was charged with DCM (12 L, 16 v) and(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide(0.75 kg, 1.999 mol, 1.0 eq.). To the suspension, MeOH (1 L, 1.33 eq)was added and resulting suspension was heated to IT=35-40° C. WhenIT=35° C., a solution of TsOH.H₂O (0.38 kg, 1.999 mol, 1 eq.) inmethanol (0.5 L, 0.67 v) was added over 10 min while maintainingIT=35-40° C. After the addition was complete a clear solution was formedwhich was then filtered through 0.45 μm filter paper. The filtrate wasdivided in to two halves and transferred into two 22-L three-neckedround-bottomed flasks with a mechanical stirrer, a thermocouple, areflux condenser and a nitrogen inlet. Each solution was stirred atIT=35-40° C. and water (32 g, 2 mol, 1 eq.) was added. The clearsolutions were then diluted with heptane (6 L, 8 v) over 15 min whilemaintaining IT=35-40° C. The resulting suspensions were allowed cooledto IT=20-25° C. over 3 h and allowed to stir at the same temperature for18 h. The solid was collected by filtration using a Buchnner funnel witha cloth filter and 20-L glass receiver under vacuum. The solids werewashed with heptane (3×2.7 v). The damp solid was dried under vacuum at30-35° C. with a slow N₂ bleed for 72 h to a constant weight to give1.06 kg of the desired product as a white solid. The material wasanalyzed and the results indicated 98.3% area HPLC purity with 7.7 ppmPd. This material was used for ball milling.

¹H NMR {DMSO (d₆)}: δ (ppm) 10.80 (br, H, NH), 8.38 (two s, 1H, CH),8.16 (two s, 1H, CH), 7.48-7.58 (m, 5.4H), 7.21-7.28 (m, 2.5H), 7.13 (d,J=7.8 Hz, 2H, CH), 6.80 (br, 3H, TsOH, H₂O), 5.0 (s, 0.8H), 4.80 (s,1.2H), 3.20 (s, 1.6H) 2.91-2.96 (m, 2.4H), 2.50-2.94 (m, 5H), 2.29 (s,3H, CH₂), 2.27 (s, 3H, CH₃)

¹³C NMR {DMSO (d₆)}:}: δ (ppm) 171.5 (C), 165.9 (C), 154.0, 152.9,149.8, 147.7, 139.2, 138.8, 134.2, 128.8, 126.3, 126.1, 125.0, 124.8,122.9, 120.0, 119.5, 118.0, 111.3, 42.4 (CH₂), 39.2 (CH₂), 30.5 (CH₂),23.8 (CH₂), 21.3 (CH₃) & 8.0 (CH₃)

DSC: 176.1° C.

TGA: 0.2652@ 240° C.

HPLC: 98.3% area (R_(T)=11.52 min)

LC-MS: 376 amu (MW. 376.17)

KF: 3.72%

Pd: 6.5 ppm

Heavy Metals: <20 ppm

Residue on Ignition: 0.1%

C₂₈H₃₂N₃O₇: Calcd. C, 61.58%; H, 5.52%; N, 7.43%

Found C, 61.49%; H, 5.53%; N, 7.46%

The resulting solid was filtered to collect the title compound; XPRD oftitle compound depicted in FIG. 12. High intensity peaks include thoseoccurring at 5, 10.5, 11.25, 14.5, 16.5, 18, 19, 19.5 and 22.5 2θ.

Example 12(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideTosylate (Anhydrate): Large-scale process

The reaction forming the title compound can be processed in multiple,e.g. eleven 150-g batches and one 25-g batch, and combined during thefinal filtration. Described below is a representative 150-g run.

A 72-L, four-neck, round-bottom flask was equipped with a refluxcondenser, an overhead, mechanical stirrer, a temperature probe with atemperature controller, and a nitrogen inlet adapter. The reactor wascharged with(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acryl-amide,prepared as described in Example 10E, (150 g) and tetrahydrofuran (40kg). The reaction mixture was heated to 60° C. to partially dissolveAPI. p-Toluenesulfonic acid (80 g, 0.42 mol) was added to the reactor asa solution in tetrahydrofuran (400 mL). The reaction was aged for 1 h at60° C. Heptane (10 kg) was then added and the internal temperaturedecreased to 51° C. The reaction mixture was heated back to 60° C. andthen allowed to cool to ambient temperature overnight. The white solidwas collected via filtration through Sharkskin filter paper and combinedwith all other runs affording 3377 g of wet solid. The solid was driedat 50° C. in a vacuum oven for 48 hand combined with ten additional150-g batches and one 25-g batch to afford 2119.3 g of product. Thesolid was then milled in 12-g batches using a Fritsch Pulverisette6-ball mill in a 240-mL bowl with 150 agate balls at 200 rpm for 1 min.The milled product was then blended in a large beaker affording 2134 gof product (87%). The ¹H NMR and ¹³C NMR spectra of the product wereconsistent with the assigned structure. ESI MS: m/z 376[C₂₂H₂₁N₃O₃+H]+.HPLC analysis (Method A): 98.8% (AUC), t_(R)=13.1 min. XRPD spectradepicted in FIG. 13.

Example 13(Z)—N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide

Scheme IV depicts one route to a cis-isomer of Compound A:

Example 14 Pharmocokinetic Studies

Male rats were used for the study of both milled and unmilled salt formsof Compound A and compared to the free base (i.e. compound A). All drugforms were dosed in the same vehicle (80% PEG400). Table 3 reports thepharmacokinetic parameters of Compound A and its mesylate and tosylatesalts in male rats following a single oral dose in 80% PEG400. Drugexposure, as measured by Cmaxor AUC, of the milled drug and the varioussalt forms increased by 3 to 8 fold when compared to the free baseunmilled form.

TABLE 3 Particle Dose Cmax AUClast Half life AUCinf Study_no Batch Saltstate (mg/kg) (ng/ml) (ng*hr/ml) (hr) (ng*hr/ml) 460180 4 Free baseUnmilled 30 421 1159 2.0 1255 460266 9 Free base Milled 30 1974 6370 5.56568 460266 10 Sulfate Unmilled 30 1940 9477 5.7 9966 460266 11 TosylateMilled 30 1613 9455 3.5 9509 460266 12 Mesylate Milled 30 884 5769 5.06039

Example 15 Solubility in Cyclodextrins

An intravenous formulation and solubility study was conducted to comparemilled free base (Compound A) and the milled tosylate monohydrate saltof Compound A in cyclodextrins. The solubilities are shown in Table 4.At cyclodextrin concentrations of 10%, the solubility of the tosylatemonohydrate of Compound A milled was approximately 10 fold higher thatthe free base milled form in sulfobutyl ether beta-cyclodextrin. Theoverall results suggest a dual mechanism of solubilization includingboth complexation with the cyclodextrin core and ion pair formationafforded by the tosylate salt form and not by the free base.

TABLE 4 Cyclodextrin Solubility (μg/ml) in: concentration HydroxypropylSulfobutyl Ether Drug form (%) Beta-Cyclodextrin Beta-Cyclodextrin Freebase milled 10 9^(a)-20^(b) 7 Tosylate 10  26 67 monohydrate milled Freebase milled 40 Not determined Not determined Tosylate 40 370 4003monohydrate milled ^(a)Procedure consisted of evaporating anethanol/water/DMA blend of the cyclodextrin and API and reconstitutingwith water ^(b)Shake-flask technique

Example 16 Mouse Infection Model

Compound A (free base) unmilled, and the tosylate salt of Compound Amilled were tested in the same mouse thigh abscess model followingsingle oral doses in 0.5% carboxymethyl cellulose. Mice (6 per dosinggroup) were rendered neutropentic, and the thigh of each mouse wasinjected with 1×10⁵ colony forming units (CFU) of Staphylococcus aureusATCC 29213. Two hours post infection, mice were dosed with the drugorally, and at 6 hours post dose, bacterial viable counts wheredetermined from the thighs of all the mice. Efficacy was calculated asthe average change in CFU/thigh compared to time 0 (time of dosing)controls. The results are presented in Table 5.

TABLE 5 Study number HH-018 Study number HH-023 Average Change AverageChange logCFU/thigh logCFU/thigh Oral Dose Free base Tosylate (mg/kg)unmilled SD milled SD 0 1.74 0.37 2.1 0.33 0.3 1.6 0.08 1.31 1.89 1 1.350.16 1.08 0.25 3 0.3 0.67 −0.42 0.19 10 0.36 0.65 −0.62 0.5 30 −0.290.06 −0.77 0.78

These data were then fitted with a standard Inhibitory Effect SigmoidEmax Model using WinNonLin v. 5.1. and the results are shown in Table 6and FIG. 14. The results show that the milled tosylate salt form was 3to 4 times more efficacious than the free base unmilled form as seen inthe static dose (dose required to reach 50% (ED50) and 80% (ED80)efficacy). This result is consistent with the increased exposure of thetosylate milled form vs. the free base unmilled form in thepharmacokinetic study (Table 3).

TABLE 6 Static dose ED50 ED80 Drug form (mg/kg) (mg/kg) (mg/kg) Freebase unmilled 10.5 4.0 8.8 Tosylate milled 2.4 1.3 3.4

Example 17 Oral Pharmocokinetic Study in Male Rats

Compound A (free base) and B (toslyate monohydrate salt of compound A)was administered to male rats, strain CD®, via oral gavage using 1%Poloxamer 407 or 0.5% carboxymethyl cellulose (CMC) as the vehicle(Table 1). All doses were calculated as free base equivalents.

TABLE 7 Experimental design Dose Number of Group Test Article Vehicle(mg/kg) Males 1 B 1% Poloxamer 407 10 12 2 B 0.5% CMC 10 12 3 B 0.5% CMC30 12 4 B 0.5% CMC 75 12 5 A 1% Poloxamer 407 10 12 6 A 0.5% CMC 10 12 7A 0.5% CMC 30 12 8 A 0.5% CMC 75 12

For each dosing group blood was collected from 3 animals at each of thefollowing time points: 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 24, and 48hours post dose. Plasma was prepared using standard techniques withsodium heparin as anti-coagulant. The Compound A and B plasmaconcentrations were determined using a GLP-validated LCMS/MSbioanalytical method. Pharmacokinetic analyses were performed using PKSolutions 2.0™ or WinNonLin 5.1.

The time-concentration profiles are shown in FIG. 15, and calculatedpharmacokinetic parameters are shown in Table 8. From these data it isapparent that the tosylate monohydrate form (B) is consistentlyassociated with greater values for Cmaxand AUClast, irrespective of thevehicle, than the free base (A). For both these pharmacokineticmeasures, the plasma levels were between 2 to 3 times higher for thetosylate monohydrate form than for the free base.

TABLE 8 Mean pharmacokinetic parameters for AFN-1252 following oraladministration in male rats Ratio of tosylate Dose Cmax AUClastmonohyrate/free base (mg/kg) Test Article Vehicle (ng/ml) (hr*ng/ml)Cmax AUClast 10 AFN-12520000 0.5% CMC 163 980 AFN-12520301 0.5% CMC 3472044 2.1 2.1 AFN-12520000 1% Poloxamer 407 157 883 AFN-12520301 1%Poloxamer 407 348 2663 2.2 3.0 30 AFN-12520000 0.5% CMC 176 1624AFN-12520301 0.5% CMC 434 3680 2.5 2.3 75 AFN-12520000 0.5% CMC 246 2373AFN-12520301 0.5% CMC 595 4256 2.4 1.8

REFERENCES

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually incorporated by reference. In case of conflict, the presentapplication, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

1. A compound of formula I:

wherein n is a fractional or whole number between about 1 and about 1.5inclusive; m is a fractional or whole number between about 0 and about 3inclusive; X is selected from the group consisting of H₂SO₄, HSO₃R′,HSO₃Ar, H₃PO₄, HCl, HBr, CF₃CO₂H, and Cl₃CO₂H; R′ is alkyl; and Ar isaryl.
 2. The compound of claim 1, wherein the compound is in acrystalline form.
 3. The compound of claim 1, wherein n equals 1, mequals 1 and X is HSO₃Ar.
 4. The compound of claim 1, wherein n equals1, m equals 0 and X is HSO₃Ar.
 5. The compound of claim 1, wherein Ar isbenzene or toluene.
 6. The compound of claim 1, wherein the compound isan anhydrous p-toluenesulfonic salt.
 7. The salt according to claim 6with characteristic peaks in the powder X-ray diffraction pattern atvalues of 2θ as depicted in FIG.
 13. 8. The compound of claim 1, whereinthe compound is a mono, di, tri or fractional hydrate form of ap-toluenesulfonic salt.
 9. The salt of claim 8, wherein the monohydrateform has characteristic peaks in the powder X-ray diffraction pattern atvalues of 20 substantially the same as depicted in FIG.
 12. 10. Thecompound of claim 1, wherein the compound is a hydrochloric acid salt.11. The salt of claim 10, wherein said salt has a powder X-raydiffraction pattern substantially that same as that depicted in FIG. 3A.12. The compound of claim 1, wherein the compound is a methane sulfonatesalt.
 13. The salt of claim 12, wherein said salt has a powder X-raydiffraction pattern substantially that same as that depicted in FIG. 4.14. The compound of claim 1, wherein the compound is a benzenesulfonicsalt.
 15. The salt according to claim 14, wherein said salt has a powderX-ray diffraction pattern substantially that same as that depicted inFIG.
 5. 16. The salt according to claim 15, wherein said salt has apowder X-ray diffraction pattern substantially that same as thatdepicted in FIG.
 6. 17. The compound of claim 1, wherein the compound isa sulfate salt.
 18. The salt according to claim 17, wherein said salthas a powder X-ray diffraction pattern substantially that same as thatdepicted in FIG.
 8. 19. A compound selected from the group consistingof:(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidehydrochloride;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidehydrobromide;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidesulfate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidemethane sulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamideethane sulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide2-hydroxyethanesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide4-methylbenzenesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide4-methylbenzenesulfonate monohydrate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidebenzenesulfonate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidephosphate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidetrifluoroacetate;(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamidetrichloroacetate;(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonicacid; Calcium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;Magnesium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;Disodium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate;Dipotassium(E)-6-(3-(methyl((3-methylbenzofuran-2-yl)methyl)amino)-3-oxoprop-1-enyl)-2-oxo-3,4-dihydro-1,8-naphthyridin-1(2H)-ylphosphonate.20. (canceled)
 21. A composition comprising the compound of claim 1 anda pharmaceutically acceptable excipient. 22.-28. (canceled)
 29. A methodof treating a subject with a bacterial illness comprising administeringto the subject the pharmaceutical composition of claim
 21. 30.-33.(canceled)
 34. A method of disinfecting an inanimate surface comprisingadministering to the inanimate surface a compound of claim
 1. 35.-42.(canceled)